Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy. DMD, which affects 1 of 3500 new-born boys, is a severe x-linked muscle wasting disease caused by nonsense and out of frame mutations of the human DMD gene. DMD patients typically require a wheelchair by age 10 to 12 and die in their late teens to early thirties.
DMD is caused by mutations in the dystrophin gene that preclude the synthesis of functional protein. The dystrophin gene is one of the largest genes in the human genome containing 79 exons spanning more than 2.3 million base pairs. Generally, a mutation in any of the exon portions of the dystrohin gene that result in changes in the reading frame by removal of an entire exon or exons or duplications of one or more exons, or introduces a stop codon, have the potential to disrupt production of functional dystrophin, which may result in DMD.
In recent years, research has shown that therapies based on antisense oligonucleotides (AOs) may provide a promising treatment of genetic related diseases including DMD. In particular, antisense oligonucleotides (AOs) have been developed that exhibit specificity to targeted gene expression. For instance, antisense oligonucleotides have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. By skipping out-of-frame mutations of the dystrophin gene, the reading frame can be restored and a truncated, yet functional, Becker-like dystrophin protein is expressed.
Studies in human cells in vitro and in animal models of the disease in vivo have proven the principle of exon skipping as a potential therapy for DMD. Initial clinical trials using two different AO chemistries (phosphorodiamidate morpholino oligomer (PMO) and phosphorothioate-linked 2′-.beta.-methyl RNA (2′OMePS)) have recently been performed, with encouraging results. Restoration of dystrophin expression in the TA muscle of four DMD patients injected with a 2′OMePS AO to exon 51 has been reported by van Deutekom et al.
One critical factor determining the effect of targeted removal of specific exon to restore dystrophin reading frame is the identification of specific antisense oligonucleotides Since antisense oligonucleotides normally have a sequence of only 20-30 nucleotides, there can be hundred or more potential candidate AOs for targeting each exon dystrophin exon. Practically, many antisense oligonucleotides do not produce efficient exon skipping. Therefore, to effectively remove an exon for the restoration of dystrophin reading frame for the treatment of DMD, antisense oligonucleotide targeting each human dystrophin exon needs to be screened and selected. However, there has been no roles to apply for the selection of most effective AO targeting individual exon. Further more, all previous arts identifies AOs which only show exon skipping effect in cell culture, which is hugely different from the final targeting cells of muscle fibers in patients of DMD. The biological effect of individual AO selected in cell culture could significantly different from the potential effect in muscle in vivo.
Accordingly, there still exists a need for improved antisense oligonucleotides which cause efficient exon skipping in targeted exons of the dystrophin gene.